Abstract

Plants synthesize a vast array of natural products, collectively known as secondary metabolites, of which flavonoids constitute an important group. Flavonoid compounds are ubiquitous in Nature and play important roles in plant physiology, development and the plant's interaction with its environment. They contribute to flower color, ultraviolet protection, cell signalling and defence against plant pathogen attack, in addition to their potential benefits to mammalian organisms. In most cases, structure-activity relationships have been observed and, therefore, modification of the substitution pattern of certain compounds may enhance the quality and/or performance of plants through metabolic engineering. Chrysosplenium americanum , a semi-aquatic weed, elaborates a variety of highly methylated flavonoids that are synthesized by regio-specific enzymes, and are believed to protect the plant against microbial attack. Several of the substitution reactions involved in the proposed pathway of their biosynthesis previously have been elucidated. One of the enzymes involved at the branchpoint of this pathway, the flavonol 6-hydroxylase (F6H) has been the primary focus of this research. The novelty of this enzyme lies in the fact that it is the only reported {460}-ketoglutarate-dependent dioxygenase that introduces a phenolic hydroxyl group on the flavonoid ring; more specifically at position 6 of trimethylquercetin, which is subsequently methylated. The dioxygenase nature of the enzyme has been established and the hydroxylation reaction has been characterized at the biochemical level, including its physicochemical properties and kinetic mechanism. The F6H protein was purified to near homogeneity in its functional state, through an original strategy involving conventional chromatography and affinity columns, and partial amino acid sequences of the purified protein were obtained. The microsequence information obtained was used as a guide in primer design for the cloning of the F6H cDNA from a C. americanum library that was screened using a PCR-based strategy. A cDNA fragment was subsequently isolated and cloned and its gene product was characterized at the biochemical and molecular levels. The protein predicted from the F6H cDNA clone contains the conserved motifs for this class of enzymes, as well as the microsequences obtained from the purified plant protein. In addition, the clone exhibits F6H activity when expressed in both prokaryotic and eukaryotic expression systems. At the molecular level, F6H is present as a single copy in the C. americanum genome, and contains two introns of approximate length and position comparable to a particular class of dioxygenases, suggesting an evolutionary relationship. (Abstract shortened by UMI.)